The LONG-TERM goal of our research is to study and analyze the structure and function of the factor V molecule in order to understand key mechanisms involved in maintaining hemostasis in the human body. In order to understand these functions, it is necessary to implement the SHORT-TERM goal of identifying the amino acid residue(s) of factor V that interact with the blood coagulation factor prothrombin (flla), during prothrombinase complex assembly and function. The specific aims of the project are stated as follows: 1) To identify the specific amino acid residue(s) of the carboxyl-terminus of the factor Va heavy chain that interact with prothrombin during prothrombinase complex formation. 2) To identify the individual amino acid(s) of factor Va that interact with prothrombin and factor Xa required to drive meizothrombin formation. 3) To identify specific acidic amino acid(s) of the factor Va heavy chain that are involved in prothrombinase complex formation. To obtain our data, factor V mutants will be constructed by PCR-based methods and purified plasmids will be transiently transfected into COS-7L cells in order to obtain recombinant proteins that will be anayzed by SDS-PAGE analysis and Western Blotting. Proteins will be screened for clotting activity in one-and two-stage clotting assays. Kinetic analyses will be done by measuring thrombin formation.